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2. REVIEW OF LITERATURE
Fermentation is process where microbes oxidise complex organic compounds like
carbohydrate into simple substances where organic acids act as electron acceptor.
The microbes digest the food substrate with its enzymes, increase the flavour, aroma, and
texture, make the food edible, enrich the food with vitamins, essential amino acids and above
all preserve food naturally. Fermented foods are produced across the world using various
techniques, raw materials and more. However, they fall in four categories namely alcoholic,
lactic acid, acetic acid and alkaline fermentation (Soni and Sandhu, 1990). Alcoholic
fermentation involves the production of ethanol as the end while lactic acid and acetic acid
fermentation produce respective acids as end products. The alkaline fermentation are not well
known but widely consumed in South east and African countries. In alkaline fermented foods,
the proteins are broken into amino acids and peptides releasing ammonia during fermentation
resulting in alkaline pH of the food which is achieved spontaneously by mixing bacteria,
especially Bacillus subtilus. A typical example for alkaline fermented food is Japanese natto
(Wang and Fung, 1996). Among these, lactic acid fermentation is widely usedfor preservation
of foods. This process is acheived by lactic acid bacteria which includes Lactobacillus,
Lactococcus, Pediococcus, Enterococcus, Leuconostoc, Weissella, Aerococcus,
Carnobacterium, Oenococcus, Sporolactobacillus, Teragenococcus, and Vagococcus.
Because of their presence in food and contribution of healthy microflora for animals, they are
‘generally recognised as safe’ (GRAS) (Donohue, 2004). LAB also known as “Lactics” are
often associated with plants (Corsetti et al., 2007; Chen et al., 2010), animals (Audisio et al.,
2011), meat (Pringsulaka et al., 2012), fermented products and often used as starter culture in
manufacture of various dairy products like cheese, curd and yogurt.
2.1 Approaches to Identify Lab
The bacteria were identified by their phenotypic characters like morphology, mode of
glucose fermentation, growth at various temperatures, pH, salt concentrations, carbohydrate
fermentation pattern, cell wall protein or whole cell protein analysis. However, these methods
are not accurate as the phenotypic characters depend on the environmental conditions which
are not reproducible. Thus, additional genotypic characterisations of isolates are also essential
to identify the organisms. Certain organisms that exhibit closely related phenotypic
characteristic feature may be well differentiated using certain techniques like RAPD, PFGE,
RFLP, DGGE and TGGE.
5
2.1.1. Randomly Amplified Polymorphic DNA (RAPD) PCR
The RAPD-PCR technique is widely used technique to discriminate closely related
organisms in which the polymorphic DNA was amplified using arbitrary primers. One
advantage of RAPD is that it does not require any knowledge of DNA sequence and amplified
product. This method is now been widely used to differentiate closely related organisms in
large number of isolates (Van Reenen and Dicks, 1996; Rossetti and Giraffa, 2005;
Albesharat et al., 2011; Bourouni et al., 2012). If there is any mutation in primer binding site,
the pattern of RAPD-PCR will be changed resulting in non-reproducibility hence it has to be
performed under controlled conditions.
2.1.2. Pulse Field Gel Electrophoresis (PFGE)
This is another fingerprinting technique which is used to distinguish closely related
organisms where DNA was subjected to electrophoresis under constant voltage with a change
in directions of 60 degree at regular interval. The DNA of higher molecular weight will
slowly realign with their charge when the field directions are changed that results in effective
separation. Unlike RAPD, this technique requires specialised instrument where standard
electrophoresis was run under constant voltage in three directions at regular interval and this
technique is laborious as it takes longer time to separate large sized DNA. Certain studies
have used this technique to identify the distinct isolates of same species (da Cunha et al.,
2012; Gonzalez-Arenzana et al., 2012). This technique gives variation that is more accurate in
the sub-typing of the organisms.
2.1.3. Restriction Fragment Length Polymorphism
In this technique, the variation in homologous DNA was explored by digesting DNA
with restriction enzymes which was separated according to their size by electrophoresis.
However, the process is cumbersome as it is time consuming and requires large amount of
DNA hence terminal restriction fragment length polymorphisms (T-RFLP) was developed. In
this method, a segment of DNA was amplified using fluorescent labelled primers following
with restriction digestion with enzymes and electrophoresis in polyacrylamide gel
electrophoresis. As the size is determined using fluorescent detector, only terminal fragments
are detected while other fragments were ignored hence this technique was used for detection
of LAB from a mixed community (Nieminen et al., 2011; Bokulich and Mills, 2012).
2.1.4. Temperature and Denaturing Gradient Gel Electrophoresis (TGGE And DGGE)
The TGGE is a method that separate molecules like nucleic acid by their melting
behaviour while DGGE separates the molecule with the aid of denaturing agents. Unlike other
methods, this is more sensitive since DNA with single nucleotide variation can be separated
6
very efficiently. A gene was amplified by PCR methods which give single band in normal
agarose gel electrophoresis will exhibit variation in DGGE because of the variations in
nucleotide sequence. This method was widely used to detect LAB from various sources like
cheese (Gori et al., 2012), milk products (Bao et al., 2012) and more. The survival rate of
LAB in intestine which was isolated from silage had been compared using DGGE (Han et al.,
2012). DGGE was widely used method as it is simple and inexpensive that does not require
specialised apparatus while TGGE required dedicated apparatus hence not widely used.
2.1.5. Phylogenetic Analysis
The 16S rRNA, a well conserved universal marker gene is the most commonly
exploited target for identification of organisms. The gene has constrained function established
in early stage of evolution which are relatively unaffected by environmental factors hence
used as a vital tool in the the study of evolutionary relationship of organism and identification.
However, it has limitations as it is well conserved with limited resolution that makes difficult
to differentiate very close organisms like Lactobacillus plantarum and Lact. pentosus. As the
copy number varies with bacterial species, it makes additional difficulty to represent certain
bacterial strains (Mohania et al., 2008). Amlified restriction digestion of ribosomal DNA is
RFLP of 16S rRNA gene product where 4 mer restriction digestion enzymes like AluI, HaeIII
are used to digest the gene product (Rodas et al., 2003). Similarly intergenic space region
(ITR) has also been used to determine the phylogenetic relationship of organisms by
comparing with prevailing sequences (Zavaleta et al., 1996). However, these could not
differentiate strains having high degree of relatedness but could differentiate species. Since it
was very difficult to identify closely related organisms, nested multiplex PCR was used to
identify them with other genes like recA (Torriani et al., 2001). Recently, various methods
like FTIR (Samelis et al., 2011), MALDI-TOF MS (Duskova et al., 2012) were introduced to
identify and cluster LAB. As whole cellular components were evaluated with FTIR and
MALDI-TOF MS, it gives complete information of phenotypic and genotypic properties. The
FTIR was used to differentiate between pathogenic and non-pathogenic Listeria and
Staphylococcus (Lamprell et al., 2006; Rebuffo-Scheer et al., 2008) while MALDI-TOF MS
was used to discriminate food borne bacteria (Mazzeo et al., 2006).
2.2. Lactic Acid Bacteria in Fermentation
Fermented foods are from plant or animal source which is processed by fungi or
bacteria that thrive over on the surface of the source. LAB are isolated from various
fermented products kimchi, doenjang, dongchimi (Lim and Im, 2009), kallappam, koozh,
morkuzhambu (Kumar et al., 2010), appam batter, vegetable pickle (Jamuna and Jeevaratnam,
7
2004a). Many new species like Leuc. kimchii (Kim et al., 2000), Leuc. inhae (Kim et al.,
2003), Weissella koreensis (Lee et al., 2002), Lact. kimchii (Yoon et al., 2000) are explored
from kimchi hence fermented food has diverse group of organisms. They are widely
distributed in nature and have a strong capability to survive in any environmental conditions
(Liu et al., 2011b).
Fermented foods can be classified as cereal based, vegetable based, vegetable/fruits
based, fish based, meat based of which cereal based fermented food are widely popular in
Asian countries and are extensively studied. Lactobacillus is the predominate organism in
theis type of fermented foods (Table 2.1) that modifies the organoleptic properties of the
fermented foods (Rathore et al., 2012). The organic acid produced by them has a role in
preservation and gives a special taste to the food. The diacetyl compounds produced during
metabolism gives a unique flavour to the food (Liu et al., 2011b). During the process of
fermentation LAB produce vitamins like riboflavin, thiamine, folic acid, etc., and enhance the
digestibility (Ghosh and Chattopadhyay, 2011) and increase the free amino acid content in the
food (Ding et al., 2009) theremy increasing its nutritional value.
In addition to fermented foods, lactic acid bacteria also play a vital role in
fermentation of medicine which was evidenced by isolation of Lact. acidophilus Kanjika,
fermented rice which is used as food and also as medicine (Reddy et al., 2007). Ayurvedic
medicine kutajarista a traditional fermented decoction of Holarrhena antidysentrica is widely
used for the treatment of various diseases like indigestion, amoebic dysentery, diarrhoea,
piles, intestinal parasite infestation and problems, fever, (Sekar and Mariappan, 2008) and
Lact. plantarum isolated from this decoction ameliorates the cellular damages caused by
Aeromonas veronii (Kumar et al., 2011a). Thus, fermentation not only enhances the nutritive
and but also the medicinal value of food making them as functional foods.
The functional foods are processed food fortified with health benefit components like
vitamins, flavones and a well-known example is addition of iodine to table salt. Fermentation
plays a major role in formulation of functional foods. Soymilk fermented with probiotic
strains increase free amino acid contents, vitamin B6, γ-aminobutyric acid, isoflavone (Li
et al., 2012). The antioxidant activity of the LAB fermented soymilk was also higher than that
of unfermented soymilk (Wang et al., 2006) which was because of changes in conjugation of
flavone and soyasaponins in soymilk (Hubert et al., 2008). In cereals fermented with
Lact. rhamnosus and Saccharomyces cerevisiae, the total phenolic content and antioxidant
activity was increased (Dordevic et al., 2010). Not only in fermented milk and cereals but
fermented fruits also have significant increase in antioxidant property which also inhibits
8
intestinal glucose and sugar uptake enzymes (Wu et al., 2011) produced during fermentation.
Ankolekar et al., (2011) has observed a significant increase with antioxidant activity,
-glucosidase and angiotensin converting enzyme inhibitors in the fermented apple juice.
LAB possesses many enzymes like polyphenol oxidase, which modify the phenolic
content in the food thereby increasing the functionality. By successive cleavage, gallatonines
were converted into gallic acid, while flavanol glycosides like kaempferol and quercetin were
converted into aglucones and bioactive polyphenol (Duckstein et al., 2012; Santos et al.,
2012) which has higher antioxidant and antimicrobial activity. Recently dihydrodaidzein
racemase has been identified in Lactococcus which is involved in conversion of daidzein, a
phytoestrogen, into equol which has beneficial effects in human (Shimada et al., 2012).
Certain other enzymes responsible for metabolism of phenolic content were discussed by
Rodriguez et al., (2009). Further characterisation of these enzymes will help in improvement
of food quality and in development of functional foods.
2.3. Lactic acid bacteria and its metabolites
Lactic acid bacteria are known to produce several metabolites that are beneficial for
humans and sometime detrimental. One of the well-known end products is lactic acid which is
used as preservative. Besides lactic acid, they also produce variety of compounds like
diacetyl, acetoine, butanediol, flavone, organic acids and various volatile components which
depend on the sources of fermentation. During fermentation process, they metabolise certain
flavanol glycoside in the plant materials into 4-hydroxybenzoic acid, gallic acid (Duckstein
et al., 2012) that exhibit antimicrobial activity (Broberg et al., 2007) and antioxidant activity
(Duckstein et al., 2012). Ganzle et al., (2009) reviewed metabolic pathways of various
carbohydrate and their end products that exhibit antifungal activity. Some organisms like
Lact. buchneri, Lact. reuteri, and Ped. pentosaceus produce propionate and propanediol that
are industrially useful and exhibit antimicrobial activity.
9
Figure 2.1. Metabolism of plant components by LAB. Adopted from Duckstein et al.,
(2012) with permission copyright © 2012.
10
Table 2.1: Certain cereal based fermented foods from Asian countries
Food Cereal Organism Reference
Appam,
Kallappam
Rice Lactobacillus plantarum (Jamuna and Jeevaratnam,
2004a),
(Kumar et al., 2010)
Boza barley,
oats, millet,
maize,
wheat or
rice
Lactobacillus plantarum
Enterococcus faecium
Leuconostoc lactis
Leuconostoc mesenteroides
subsp. dextranicum
Pediococcus pentosaceus
Lactobacillus fermentum
Lactobacillus paracasei
Lactobacillus pentosus
(Todorov and Dicks,
2005a)
(Todorov and Dicks,
2005b)
(Todorov, 2010)
(von Mollendorff et al.,
2006)
(Petrova and Petrov, 2011)
Brem Maize Pediococcus pentosaceus
Enterococcus faecium
Lactobacillus curvatus
Weissella confuse
Weissella paramesenteroids
(Sujaya et al., 2001)
Burong Isda Rice Lactobacillus plantarum (Olympia et al., 1995)
Chili Bo Maize Lactobacillus plantarum
Lactobacillus fermentum
Lactobacillus farcimini
Pediococcus acidilactici
Enterococcus faecalis
Weissella confusa
(Leisner et al., 1999)
Dosa,
Idly,
Dhokla
Rice Lactobacillus plantarum
Pediococcus pentosaceus
Lactococcus lactis
Enterococcus faecium
Leuconostoc mesenteroides
(Iyer et al., 2011)
(Sawale and Lele, 2010)
(Vijayendra et al., 2010)
Injera Wheat,
Barley,
Corn, rice
Leuconostoc mesenteroides,
Streptococcus faecalis
Pediococcus spp.
Lactobacillus spp.
(Gashe, 1985)
(Nigatu et al., 1998)
Kanjika Rice Lactobacillus acidophilus (Reddy et al., 2007)
Khanomjeen Rice Lactobacillus plantarum (Oupathumpanont et al.,
2009)
Koozh Millet Weissella paramesenteroides
Lactobacillus plantarum
Lactobacillus fermentum
(Kumar et al., 2010)
Miso Rice/Wheat Enterococcus durans
Enterococcus faecium
Lactococcus spp.
(Onda et al., 2002)
Puto Rice Leuconostoc spp.
Enterococcus faecium
(Kelly et al., 1995)
(Shibata et al., 2007)
Sourdough Wheat Many Lactobacillus spp. (Gobbetti, 1998)
11
2.3.1. Antimicrobials
2.3.1.1. Organic Acids
When the carbohydrates are fermented, they produce wide variety of organic acids that
will reduce the pH of the environment resulting in reduction of unfavourable organisms. Two
major acids, namely lactic acid and acetic acid are produced by LAB that exhibit
antimicrobial activity at low pH than at neutral pH among which acetic acid is more effective
as they control the growth of yeast, molds and Bacilli (Reis et al., 2012). The dissociated
lactic acid in cytoplasm causes acidification resulting in failure of proton motive force. A
synergistic effect of acetic acid and lactic acid was observed at reduced concentration when
compared to individual concentration. In addition to antimicrobial activity, acetic acid also
contributes for the aroma (Reis et al., 2012). Propionic acid though produced in low
concentration is known to interact with cell membrane and neutralise the electrochemical
proton gradient but not as like lactic acid which also inhibit amino acid uptake (Reis et al.,
2012). Certain Lactobacillus spp., and Pediococcus are known to produce 2-pyrrolidone-5-
carboxylic acid that exert antimicrobial activity against spoilage bacteria (Yang et al., 1997)
whose mode of action may also similar to that of lactic acid (Ouwehand and Vesterlund,
2004) but was not as efficient. Additional to these organic acids, they produce benzoic acid
(Niku-Paavalo et al., 1999), 5-hydroxyl ferulic acid (Ou and Kwok, 2004; Knockaert et al.,
2012), 3-phenyllactic acid (Rodriguez et al., 2012), 3-hydroxydecanoic acid (Broberg et al.,
2007), hydroxyl fatty acid (Sjogren et al., 2003) and more that exhibited antimicrobial
activity. Reuterin, chemically β-hydroxypropionaldehyde produced by Lact. reuteri is a non-
peptide broad spectrum antimicrobial compound synthesised from glycerol exhibit
antimicrobial active against prokaryotes and eukaryotes (Chung et al., 1989). Reuterin as
analog of D-ribose, inhibit B1 subunit of ribonucleotide reductase and thioredoxin suggesting
its inhibition of sulfhydryl enzymes (El-Ziney et al., 2000). Some of the widely studied
organic acids that exhibited antimicrobial activity are given in Table 2.2.
2.3.1.2. Bacteriocins
Bacteriocins are diverse group of modified or unmodified antimicrobial peptide
extracellularly secreated by bacteria which are protected by dedicated immune system (Rea
et al., 2011). Since the discovery in 1925 by Gratia, search of bacteriocins from various
organisms has been intensified resulting in purification and characterisation. Though many
bacteriocins were reported from various organisms, the peptide antimicrobials from LAB
were considered to be innocuous as they are naturally associated with human and animals and
can safely be employed in food preservation and medicine (Rea et al., 2011) as they are active
12
against diverse group of microbes including fungi (Adebayo and Aderiye, 2010). Gram
positive bacteria has gained specific regulatory mechanisms for synthesis and secreation of
bacteriocins sparing the producer strain. Bacteriocin gene operon which was usually
associated with plasmid (Daeschel and Klaenhammer, 1985; Mathys et al., 2007) encodes
specific transport system for bacteriocin, an immunity protein and the prepeptide of
bacteriocin which was secreted extracellularly after processing.
Table 2.2: Some organic acids produced by LAB that exhibit antimicrobial activity
Compound Structure
Lactic acid
Acetic acid
Propionic acid
2-pyrrolidone-5-carboxylic acid
Benzoic acid
5-Hydroxy ferulic acid
3-phenyllactic acid
Reuterin
13
Figure 2.2. Synthesis and secreation of bacteriocin by Gram positive bacteria Adopted
from Ennahar et al., (2000) Copyright© 2000
14
2.3.1.2.1 Detection of Bacteriocin
The concentration of bacteriocin are expressed as arbitrary units (AU) which is
defined as reciprocal of highest dilution exhibiting minimal inhibition. Parente et al., (1995)
have detailed various ways of screening bacteriocin activity among which agar well diffusion
is widely used because of its reliability. While screening for bacteriocins, it is essential to
eliminate the inhibition by non bacteriocin agents like bacteriophage, organic acids, H2O2 and
non-ribosomally synthesised antimicrobials like mevalonolactone and cyclic dipeptides.
Evaluation of bacteriocin preparation at the pH of 6 eliminates the effect of acids and
treatment with protease should lead to loss of activity (Moraes et al., 2010).
2.3.1.2.2. Classification of Bacteriocin
The classification of bacteriocin produced by LAB is much complicated because of
heterogeneity hence various classification schemes has been proposed. However,
Klaenhammer, (1993) classification of bacteriocin into four groups has formed a basis of all
prevailing classification. According to Klaenhammer,
Class I bacteriocins or lantibiotics are small peptides (<5 kDa) that has unusual amino
acids like lanthionine, β-methyl lanthionine (lantibiotics).
Class II bacteriocins are unmodified heat stable membrane active peptides whose
molecular weight are less than 10 kDa. These are further classified into three classes
Class IIa bacteriocin that has a conserved YGNGV sequence in N-terminal region
exhibiting antilisterial activity.
Class IIb bacteriocins are two peptide bacteriocins
Class IIc bacteriocins are thiol activated peptide whose activity was lost if disulphide
bridges are broken
Class III bacteriocins are unmodified heat labile proteins whose molecular weight are
greater than 30 kDa. These are associated with enzymatic activity.
Class IV bacteriocins are complex proteins that are associated with lipids or
carbohydrate moieties.
This classification was further refined by Cotter et al., (2005) as Class I lantibiotics
and Class II peptides which are further classified as IIa: pediocin like bacteriocins, IIb: two
peptide bacteriocins, IIc: cyclic peptides and IId: non-pediocin unmodified bacteriocins and
Class III bacteriolysin which are lytic peptides. Heng et al., (2007) and Nissen-Meyer et al.,
(2009) have further refined the classification which has sub-classified Class II bacteriocins
based on amino acid sequence and moved cyclic peptides to Class IV.
15
2.3.1.2.2.1. Class I: Post Translationally Modified Bacteriocins
The bacteriocins of this class undergo post translation modification to get lantibiotics.
This group of bacteriocins are well characterised as Nisin of this group is used as food
preservative. Because of additional post translational modifications, this group was further
classified into three groups by Rea et al., (2011)
Class Ia: Lantibiotics
The lanthionine containing antibiotics are generally less than 5 kDa with 19-30 amino
acids that undergo post translation modification to produce unusual amino acids. Lantibiotics
are further classified into type A linear lantibiotics which includes Nisin and subtilin while
type B globular bacteriocin that include mercsacidin, cinnamycin, mutacin II and lacticin 481.
The mode of action of type A lantibiotics is by membrane pore formation resulting in
dissipation of membrane potential causing cell death while type B acts as inhibitor of cell wall
synthesising enzymes.
Class Ib: Labyrinthopeptins
Labyrinthopeptins are globular hydrophobic peptide which was recently identified
from Actinomadura namibiensis (Meindl et al., 2010). This bacteriocin was distinguished by
the presence of labionins derived after modification from S-X-X-S-X-X-X-C motif by
LabKC, a bifunctional protein which posses Ser/Thr kinase and lanthionine cyclase. This
bacteriocin has a notable activity against herpes simplex virus.
Figure 2.3. Crystal structure of Labyrinthopeptins with labionins Adopted from
Meindl et al., (2010) copyright © 2010.
16
Class Ic: Sactibiotics
A circular bacteriocin, subtilosin A produced by Bacillus subtilis (Kawulka et al.,
2004) that has unusual sulphur cross linkage between Cys and -carbon of Phe and Thr was
not considered to be circular bacteriocin hence Martin-Visscher et al., (2009) has proposed to
place it in a unique class. Hence it was included in Class I bacteriocin as it was post
translationally modified to produce such linkage. Another two peptide bacteriocin thuricin CD
produced by Bacillus thuringiensis 6431was also identified to have such cysteine to -carbon
linkage (Rea et al., 2010) which does not show any sequence similarity with subtilosin A. The
genetic sequence of thuricin operon consists of two regions, one coding for bacteriocin and
another overlapping ORF of trnC and trnD that code for radical S-adenosyl methionine (SAM
superfamily protein) (Rea et al., 2010). The unusual cysteine to -carbon linkage was
produced by two [4Fe-4S] clusters of radical SAM enzyme where one involved in cleavage
reaction while another is required for generation of thioester linkage (Fluhe et al., 2012).
Figure 2.4 Structure of subtilosin A that shows unique thioester linkage Adapted with
permission from Kawulka et al., (2004) Copyright © 2004
17
2.3.1.2.2.2. Class II: Unmodified Bacteriocins
The class II bacteriocins are essentially unmodified bacteriocin consisting standard
amino acids with molecular weight <10 kDa. According to Heng et al., (2007), it is further
subdivided into Class IIa: Pediocin like bacteriocin, Class IIb: two peptide bacteriocin,
Class IIc: other unmodified bacteriocins
Class IIa: Pediocin Like Bacteriocin
A large collection of Class II bacteriocins produced by Gram positive bacteria belong
to this sub-class. It has a potential application as food preservative and possible biomedical
application as it is effective against Listeria, Staphylococcus aureus, Bacillus cereus and
Clostridium perfringens. Pediocin PA-1 is the best characterised bactericion of this class and
is the only antilisterial bacteriocin from this class used as preservative as a constituent in
AltaTM
2341. This group of bacteriocin was characterised by the conserved amino acid motif
YGNGV at N-terminal end and presence of disulphide bridge. The bacteriocin exhibits
bactericidal mode of action where the conserved region binds with IIC and IID components of
mannose permease phosphotransferase system and insertion of bacteriocin inside the
membrane forming pore that caused membrane permeabilization.
Figure 2.5. Sequence alignment of Class IIa bacteriocin sequence collected from
BACTIBASE showing conserved region
18
Figure 2.6. Mode of action of of Class II bacteriocin adopted with permission from
Ennahar et al., (2000) copyright © 2000
19
Class IIb: Two Peptide Bacteriocin
The bacteriocins of this class include plantaricin EF which has two peptides and
requires both the peptides in equal concentration to exert its activity. Till date, only 16
bacteriocins were reported since the discovery of lactococcin G (Nissen-Meyer et al., 2009).
The mode of action of this bacteriocin has revealed the permeability of target cells leading to
cell death (Nissen-Meyer et al., 2009). The gene coding for this bacteriocin are found adjacent
to each other with immunity protein and ABC transporter. Although conserved regions were
not detailed much in this class, a common GXXXG motif was essential for antimicrobial
activity (Rea et al., 2011). The peptides are active only with their counterpart though 60-70%
similarity was observed. However, the activity was observed when lactococcin G was
combined with its complimentary peptide of enterocin which exhibited nearly 88% homology
(Nissen-Meyer et al., 2009).
Figure 2.7. 3D structure of plantaricin J
Cass IIc: One Peptide Non-Pediocin Like Bacteriocin
The unmodified non-pediocin like one peptide cationic, hydrophobic bacteriocin
which does not have similarity with Class IIa bacteriocins are placed in this group. It includes
lactococcin A, enterocin B, cornobacteriocin B, acidocin 1B, mesenterocin 52B and more.
Lactococcin A was the first of this type isolated from Lactococcus lactis which also increases
permeability of membrane. This bacteriocin also binds to mannose phosphotransferase
permease embedded in the target cells and inserted inside the membrane resulting in pore
formation. Several enterococcal bacteriocins like enterocin EJ97, LA50, LB50, Q belong to
this group (Heng et al., 2007).
20
2.3.1.2.2.3. Class III: Large Bacteriocins
These bacteriocins are heat labile large proteins of molecular weight more than 10 kDa
with exception of propionisin SM1 which is heat stable. This group is further subdivided into
two groups as IIIa: bacteriolysin and IIIb: non-lytic antimicrobial proteins. Bacteriolysins are
plasmid mediated glycyl-glycine endopeptidase that hydrolyse pentaglycine cross bridges in
peptidoglycans of Gram positive bacteria resulting in cell lysis. One well studied bacteriocin
of this group is lysostaphin, a 27 kDa bacteriocin produced by Staphylococcus simulans that
exhibit antimicrobial activity against pathogenic organisms like Staphylococcus aureus and
Staphylococcus epidermidis (Heng et al., 2007). Unlike this, dysgalacticin and streptococcin
A-M57 does not kill the target organism by lysis mechanisms. Dysgalacticin produced by
Streptococcus dysgalactiae subsp. equisimilis blocked the glucose uptake targeting
PEP-dependent glucose and mannose transporter and also exhibited bactericidal effect by
increasing the membrane permeability by leaking intracellular potassium ions on
Streptococcus pyogenes (Swe et al., 2009). The structural gene of dysgalacticin was located in
plasmid and the strain lost its activity when the plasmid was cured. In addition to this, the
strain also became resistant for exogenous dysgalacticin which showed that the producer
strain had immunity protein dsyI which acted at membrane level to prevent its effect on target
cells (Swe et al., 2010).
Figure 2.8. 3D structure of lysostaphin a class IIIa bacteriocin Rendered from Protein
database PDB 1QWY.
21
2.3.1.2.2.4. Class IV: Cyclic Peptide Bacteriocins
These bacteriocins were classified as separate class because of its unique feature
(Heng et al., 2007). As like other bacteriocins, these are also ribosomally synthesised which
undergo post translational modification to form a covalent amide linkage between N and C
terminal last amino acids of mature peptides. Because of this nature, these bacteriocins are
resistant towards various proteases and are stable across wide temperature. Ever since the
discovery of enterocin AS48 (Samyn et al., 1994), the study on circular bacteriocins had been
increased that lead to the discovery of gassericin A (Kawai et al., 1998), carnocyclin A
(Martin-Visscher et al., 2008), lactocyclicin Q (Sawa et al., 2009), leucocyclicin Q (Masuda
et al., 2011). Based upon the amino acid composition this bacteriocin is further subdivided
into group IVa which is cationic peptides that include enterocin AS-48, circularin A,
uberolysin, lactocyclicin Q, carnocyclin A and garvicin ML which has high pI value
of nearly 10. Group IVb is anionic peptides that included gassericin A and butyrivibriocin
AR10.
Figure 2.9. Protein sequences of Class IV cyclic peptide showing two different subclasses
and 3D structure of Enterocin AS-48 and Carnocyclin A Adapted with permission from
van Belkum et al., (2011). Copyright © 2011
22
2.3.1.2.3. Applications of Bacteriocin
The bacteriocins produced by LAB have several striking features that made attractive
for using them in food preservation and medical purpose. They are considered as safe and
easily digested by intestinal enzymes thus do not alter the intestinal microbiota. As they exert
synergistic effect with other antimicrobials, exerting wide spectrum of inhibition against food
spoilage and pathogenic bacteria, it can effectively be used as food preservative (Jamuna
et al., 2005). Though many bacteriocin preparations were studied, only Nisin and Pediocin are
commercially available for direct use in food because of regulations in laws across many
countries. As it is cationic and hydrophobic, they build electrostatic interactions with negative
charges of bacteria and make pore on the membrane. In addition, they exhibit spermicidal and
antitumor activity (Silkin et al., 2008) (Villarante et al., 2011). Hence, the topical application
of bacteriocin in the field of medicine for treatment against various pathogens in human and
cattle are focused. Mersacidin produced by Bacillus inhibited cell wall synthesis of
methicillin-resistant Staphylococcus aureus in mice while nisin inhibited the growth of
Staphylococcus aureus in respiratory tract of rats, Clostridium tyrobutyricum, C. difficile
(Dicks et al., 2011). Nisin produced by Lactococcus lactis AMB1 and IB-367 has given
successful results against Helicobacter pylori in phase I clinical trials. Likewise, subtilosin, a
class Ic bacteriocin, isolated from Bacillus amyloliquefaciens has proved to exhibit
antimicrobial activity against Gardnerella vaginalis, bacterial vaginosis associated bacteria.
This bacteriocin was active only against pathogens sparing native vaginal Lactobacillus with
negligible cytotoxicity and exhibited spermicidal activity (Sutyak et al., 2008a; Sutyak et al.,
2008b) hence this bacteriocin can safely be used in personal care products for treatment of
bacterial vaginosis. In addition, as this bacteriocin also exhibit spermicidal activity on higher
animal models like horse, it can be used in animal models as spermicidals. This has another
advantage than nisin which also exhibited spermicidal activity (Aranha et al., 2004) as it does
not affect the native vaginal isolates while the fate of vaginal isolates upon usage of nisin is
unknown. Dicks et al., (2011) had detailed about the application of bacteriocin in treatment
against various pathogenic bacteria associated with respiratory diseases, neuroparalytic
diseases, oral and dental diseases, gastric diseases. Certain lantibiotics like duramycin also
play a vital role as anti-inflammatory agents by sequestering phosphatidylethanolamine or
inhibit angiotensin converting enzymes. However, further use of bacteriocins as medicines
need thorough investigations as they may also cause some immune responses.
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2.3.2. Exopolysaccharides
Exopolysaccharides (EPS) are metabolite of bacteria which are simple or complex in
structure and composition for protection of cells against stress like metals, phage attacks,
desiccation, for facilitation of adhesion onto substratum, for biofilm formation and more
(Farnworth et al., 2007). Depending on the composition and structure of EPS produced by the
bacteria, the texture of food is modified hence it has essential value in food industry. Among
the EPS producing bacteria, study of EPS produced by LAB has increased because their
GRAS status. Based on the composition, EPS was classified into homo-polysaccharide that
has single type of sugar unit and hetero-polysaccharide that has more than one type of sugar
unit (Jolly et al., 2002).
2.3.2.1. Homo-polysaccharides
Most of the homo-polysaccharides are synthesised from sucrose with the help of
glycosyltransferase family. Some commonly studied homo-polysaccharides are dextran,
levan, alteranan, reuteran which are synthesised by respective glycosyltransferase enzymes
namely dextransucrase, levansucrase, alteransucrase and reuteransucrase. Among these homo-
polysaccharides, dextran that has glucose with (16) glycosidic linkage as main chain and
varied degree of (12), (13) and (14) branching is widely studied. It is used for
matrix preparation of chromatographic columns, to restore blood volume, though it has side
effects in medical application (Patel et al., 2012).
2.3.2.2. Hetero-polysaccharides
It is made of several repeated units of two or more monosaccharide units or its
derivatives like N-acetyl-D-glucosamine. Synthesis of hetero-polysaccharide involves gene
cluster that include region for enzyme regulation, chain length determination,
glycosyltransferase, for export of synthesised EPS (Ruas-Madiedo et al., 2012).
2.3.2.3. Application of Exopolysaccharides
The EPS is not digested by intestinal enzymes and because of its high viscosity; it
increases the bulk faecal transit. They are digested by colonic bacteria especially
Bifidobacterium longum and produces short chain fatty acids which has many beneficial
properties in host (Hongpattarakere et al., 2012). It is also observed to alleviate
immunomodulatory property (Liu et al., 2011a), posses antitumor, antiviral activity and used
to eliminate heavy metal (Kumar et al., 2007) for lowering blood glucose level in borderline
type II diabetes (Farnworth et al., 2007). Thus, intense research in structural and functional
characterisation of EPS will enhance applicability in the field medicine.
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2.4. Application of Lactic Acid Bacteria
LAB have wide application as probiotic organisms for its multifactorial benefits to
human being and other animals. Probiotics are defined as “Live microorganisms which when
administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2002). In
early 20th
century, Metchnikoff proposed the role of gut flora in human health which was
termed as probiotics by Werner Kollath in 1953 (http://en.wikipedia.org/wiki/Probiotic).
Many organisms possessing probiotic property from various sources were reported, among
them Lact. rhamnosus GG (Lebeer et al., 2010) and Bifidobacterium (Schell et al., 2002)
strain have exhibited an effective probiotic property. World health organization (FAO/WHO,
2002) and Indian Council of Medical Research (ICMR-DBT, 2011) has suggested certain
properties which probiotic organisms should posses. According to them, the probiotic
organisms must be tolerant to acid and bile, secrete antimicrobial substances and bile
hydrolase, should produce β-galactosidase and adhere to intestinal epithelial cells. Probiotic
Lact. plantarum AS1 isolated from kallappam (Kumar et al., 2010) binds with
adenocarcinoma cells via carbohydrates and proteins (Kumar et al., 2011b) and also reduced
the development of DMH-induced cancer by antioxidant dependent mechanism (Kumar et al.,
2012b). Bhakta et al., (2010) has identified probiotic Pediococcus exhibited resistance to
heavy metals like arsenic. Several studies have reported the ability of LAB to remove metals
and certain toxic compounds from environment (Haskard et al., 2001; Halttunen et al., 2007b)
(Zinedine et al., 2005). However, removal is influenced by pH, where maximum removal
occurs at pH 4-6 while below pH 3 there is a sharp decrease in adsorption (Halttunen et al.,
2007a; Bhakta et al., 2012). Unlike other cationic metals like cadmium, lead which can bind
on the negative charges on the surface of cell membrane, the cell surface charge of LAB have
to be modified for removal of arsenic (Halttunen et al., 2007a). In addition to heavy metal
resistance, LAB is also known to degrade phytate, anti-nutritional factor (Anastasio et al.,
2010). Thus, LAB exhibiting probiotic properties with removal of heavy metals and toxic
substances may exhibit promising medical application as probiotic supplement. These
potential values of LAB have prompted to devise this study of isolation and characterisation
of LAB from south Indian fermented food source Idly batter. The antimicrobials substances
and exopolysaccharide produced by potent isolates having beneficial properties like heavy
metal resistance were characterised.